The kinetics of a biomolecule such as a protein or the interaction between biomolecules can be often investigated by a fluorescence intensity measurement in which an object to analyze is labeled with a fluorescent product or a fluorescent dyestuff. As such fluorescent dyestuff, Alexa Fluor, BODIPY FL, Cascade Blue, FITC, Oregon Green, RITC, Texas Red, TRITC, Coumarin Maleimide, Cy Dye, Dansyl Chloride, Dansyl Hydrazine and so on can be used.
A nonnatural amino acid having a functional side chain is synthesized and then introduced into a position-specific manner as the same manner as a natural amino acid or used in a peptide synthesis system to allow introduction of various functional groups without damaging the function of a protein. For example, if a nonnatural amino acid conjugated with a fluorescent substance could be introduced into the specific position of a protein, or if a fluorescent nonnatural amino acid could be applied in a peptide synthesis system, it would be expected to facilitate simple and appropriate analysis of the kinetics of a biomolecule or the interaction between biomolecules.
There is a report relating to the synthesis of a fluorescent amino acid having an acridine skeletal (Non patent document No. 1). There is disclosed a novel acridone dyestuff derivative having a characteristic lifetime of fluorescence (Patent document No. 1). The Patent document No. 1 also describes a set of different fluorescent acridone derivative dyestuffs in which the dyestuffs are characterized by their respective changes in lifetime of fluorescence, and further reports an acridone dyestuff derivative which is particularly useful in a multi-parameter analysis. The fluorescent amino acid or the acridone dyestuff derivatives reported in these documents are fluorescent substances which are suitably excited to use by ultraviolet excitation. On the other hand, there is a commercially available a compound having the BODIPY(R) (Molecular Probes) skeletal as a fluorescent substance which can be excited by a visible light and improved to be high in light stability. The compound is high in absorption coefficient and quantum yield of fluorescence to emit strong fluorescence, but the compound, which has a large side chain, is introduced into a protein to destroy the higher order structure of the protein, which makes it difficult to introduce the compound inside the protein.
For analysis of the kinetics of a biomolecule or the interaction between biomolecules, there has been used a commonly applicable measurement device such as a confocal microscope or a microplate reader using a visible argon laser as a light source. There has been recently developed a blue semiconductor laser, which has been used as a light source to provide a very compact measurement device for the analysis. The above analysis using the measurement device needs to provide a fluorescent dyestuff which can absorb efficiently the blue laser beam and has an absorption band from the ultraviolet region to the shorter wavelength visible region.
The fluorescent amino acid corresponding to a measurement device using the blue laser beam includes L-2-acridonyl alanine (acdAla) as a candidate, one of a nonnatural amino acid which has a side-chained acridonyl group as the fluorescent probe. The acdAla can relatively satisfy following conditions necessary for the fluorescent label of a protein: (1) the molecules has an absorption band and a fluorescent wavelength toward a longer wavelength side than the fluorescent amino acid such as tryptophane does; (2) the label has a high fluorescent quantum yield; and (3) the label has a small fluorescent side chain. However, the production (synthesis) of Boc-acdAla protected with Boc needs six reaction steps, some of which have a problem for the reactions to lower their respective yields. Thus, the production not only takes much expenses and time, but also brings a low yield.    [Non-patent document No. 1] Helvetica Chimica Acta., 86, 3326 (2003)    [Patent document No. 1] JP A2005-500406